The present invention relates to a novel sorbitol dehydrogenase, a microorganism for producing such an enzyme and a process for the production of a sorbitol dehydrogenase using the same. More particularly, the present invention relates to a sorbitol dehydrogenase which acts on sorbitol with high sensitivity and high selectivity, a microorganism for producing such an enzyme and a process for the production of a sorbitol dehydrogenase using the same. The present invention further relates to a method for the measurement of sorbitol using the foregoing sorbitol dehydrogenase and a reagent for the quantitative determination therefor.
D-Sorbitol is a compound which occurs in human erythrocytes and serum in a slight amount. It is known that the content of D-sorbitol is an important indication of certain diseases, particularly diabetes. It is reported that D-sorbitol is useful as a diagnostic marker for diabetes.
D-sorbitol and D-fructose have long been widely used as sweetening agents in the food industry.
Examples of the process for the measurement of sorbitol include a process involving gas chromatography, a process involving liquid chromatography, and a process involving the use of an enzyme. However, both the process involving gas chromatography and the process involving liquid chromatography require a complicated operation so that it is difficult to treat a large amount of test samples.
The process involving the use of an enzyme comprises the oxidation of D-sorbitol with a sorbitol dehydrogenase (EC 1.1.1.14) in the presence of NAD+, wherein the amount of NAD+ reduced to NADH is measured based on fluorescent intensity. This method can be easily applied to automatic: analyzers and presently finds widest application.
Examples of sorbitol dehydrogenases which have heretofore been known include those derived from animal liver such as sorbitol dehydrogenase derived from sheep liver available from Rosch Inc., and those derived from microorganisms such as an enzyme derived from Pseudomonas sp. described in xe2x80x9cEnzyme Microb. Technol.xe2x80x9d, vol. 13, pp. 332-337, April, 1991, an enzyme derived from Bacillus subtilis described in xe2x80x9cJournal of Biological Chemistryxe2x80x9d, vol. 267, No. 35, pp. 24,989-24,994, 1992, an enzyme derived from Pseudomonas sp. described in xe2x80x9cJournal of Fermentation and Bioengineeringxe2x80x9d, vol. 84, No. 3, pp. 254-256, 1997, and JP-08-033482, and an enzyme derived from Bacillus fructose described in xe2x80x9cBiosci. Biotechnol. Biochem.xe2x80x9d, vol. 63, No. 3, pp. 573-574, 1999.
However, the foregoing sorbitol dehydrogenases are disadvantageous in that they have a low substrate specificity and a high Km with respect to sorbitol. In other words, the foregoing sorbitol dehydrogenases derived from animal liver, sorbitol dehydrogenases derived from Bacillus subtilis, and sorbitol dehydrogenases derived from Pseudomonas sp. show almost the same reactivity to iditol, iditol and xylitol, and galactitol, respectively, as sorbitol. Further, all the foregoing sorbitol dehydrogenases exhibits a Km of about 10 mM or more with respect to sorbitol, which presents difficulties in the measurement of sorbitol.
On the other hand, the sorbitol dehydrogenases disclosed in JP-56-029994, U.S. Pat. No. 5,747,301, EP-728840, WO 99/20763, EP-897984 and KR-98069057 are enzymes which produce sorbose with sorbitol as a substrate and thus are different from the enzyme of the present invention.
Further, JP-06-209793, JP-06-189790, JP-01-08692, U.S. Pat. No. 5,250,420, WO 92/21775, JP-07-322897, and JP-06-109726 disclose a process for the measurement of sorbitol using a sorbitol dehydrogenase. However, JP-06-209793 and JP-06-189790 concern a process for the measurement of sorbitol using the enzyme disclosed in the above cited JP-56-029994; JP-10-108692 concerns a process for the measurement of sorbitol using the enzyme disclosed in the above cited JP-08-033482; and U.S. Pat. No. 5,250,420, WO 92/21775, JP-07-322897, and JP-06-109726 concern a process for the measurement of sorbitol using a commercially available enzyme. None of these references describes the enzyme disclosed.
Moreover, the following references describe sorbitol dehydrogenases, but none of them describes the enzyme disclosed herein. That is, EP-524517, EP-791355, WO 94/07867, U.S. Pat. No. 5,998,463, SU-1553897, WO 98/33936, and SU-1567626 refer to sorbitol dehydrogenases derived from the human body concerning the treatment of diabetes. The sorbitol dehydrogenases to be used in the processes disclosed in JP-09-037796, JP-63-146800, JP-61-005091, JP-9-2066875, and DE-3326546 are enzymes the origin of which is not specified or which are commercially available. SU-1303937 refers to a sorbitol dehydrogenase derived from tea leaves. WO 94/15942 refers to a sorbitol dehydrogenase derived from yeast. DE-2022280 discloses a process for the measurement of xylitol. All of these are quite different from the enzyme of the present invention.
It is therefore an object of the present invention to provide a novel sorbitol dehydrogenase having excellent substrate affinity and substrate specificity which can be used to measure D-sorbitol which occurs in human erythrocytes and serum in a slight amount or D-sorbitol or D-fructose contained in foods, a microorganism for producing such an enzyme, and a process for the easy production of the enzyme using such a microorganism.
The inventors made extensive studies to solve these problems. As a result, they found that a microorganism belonging to Flavimonas and Pseudomonas separated from the soil produces a sorbitol dehydrogenase having-excellent substrate affinity and substrate specificity. Thus, the present invention has been accomplished.
A first aspect of the present invention is directed to a sorbitol dehydrogenase having the following physicochemical properties.
(1) activity: the sorbitol dehydrogenase catalyzes dehydrogenation oxidation of D-sorbitol in the presence of NAD+ to produce D-fructose, and catalyzes a reverse reaction which reduces D-fructose in the presence of NADH to produce D-sorbitol and NAD+;
(2) substrate specificity: the sorbitol dehydrogenase exhibits a Vmax/Km value of about 40 or less for galactitol and about 3 or less for L-iditol when the Vmax/Km value for D-sorbitol is taken as 100, and which does not act on D-arabitol, D-mannitol, xylitol and D-glucose; and
(3) exhibits a Km value of about 6 mM or less (preferably 4 mM or less) with respect to D-sorbitol.
A second aspect of the present invention is directed, to a microorganism belonging to Flavimonas or Pseudomonas capable of producing the foregoing sorbitol dehydrogenase.:
A third aspect of the present invention is directed to a process for the production of a sorbitol dehydrogenase, which comprises culturing the foregoing microorganism capable of producing a sorbitol dehydrogenase in a culture medium, and then collecting sorbitol dehydrogenase from the culture medium.
A fourth aspect of the present invention is directed to a process for the measurement of sorbitol, which comprises adding a composition containing the foregoing sorbitol dehydrogenase to a sample to cause a reaction, and then measuring the content of sorbitol in the sample.
A fifth aspect of the present invention is directed to a reagent for the quantitative determination of sorbitol comprising the foregoing sorbitol dehydrogenase, NAD+ and a buffer.
The sorbitol dehydrogenase of the invention has excellent substrate specificity and substrate affinity, and thus is useful for the measurement of sorbitol.